1. PROJECT SUMMARY/ABSTRACT Summary The membrane-bound Ras GTPases are key regulators of diverse cellular functions. Despite decades of research, mechanisms of how Ras operates on the membrane to activate its effectors remain poorly defined. Recent high-resolution imaging studies suggest that formation of dynamic, nanoscopic lipid-protein clusters termed nanoclusters on the membrane may be critical to Ras function. Based on evidence primarily from immuno-EM, it was further hypothesized that different Ras isoforms occupy non-overlapping membrane domains to form spatially and functionally distinct nanoclusters. However, technical limitations of immuno-EM have precluded thorough analyses of the molecular compositions of the membrane domains involved in Ras clustering and how Ras interacts with these domains. To date, it is still debated whether Ras clustering and signaling take place in specialized membrane domains, and if so, what these domains are, and how their composition and structure impact Ras clustering and signaling properties. To address these questions, the PI?s lab uses superresolution microscopy (SRM), correlative SRM-EM, and high-throughput single-particle tracking (SPT) to study Ras in model cell lines. These new imaging tools allow quantitative analysis of molecular location, stoichiometry, diffusion, and interaction in live or fixed cells along with the nanoscopic cellular context. Using these tools, we identified regions of the membrane, referred to as Ras anchoring nano-domains (RANDs), that transiently trap Ras to potentially facilitate clustering. Preliminary data also suggest the presence of RANDs with diverse compositions and structures, which could play a key role in Ras regulation and account for the diverse and context-dependent cellular functions of Ras. Prompted by these initial findings, we propose to systematically analyze RANDs in composition, structure, and roles in Ras clustering and signaling in three specific Aims. First, we will define the mechanisms of Ras clustering in RANDs. We will test the hypothesis that Ras forms clusters in RANDs through HVR-dependent localization followed by G-domain mediated Ras-Ras interaction by using single-molecule FRET (smFRET) and computer simulations. Second, we will use multiplexed SRM and correlative SRM-EM to determine the molecular and structural identities of RANDs and test the hypothesis that Ras localizes to diverse RANDs depending on the biological context. Third, we will define the role of RANDs in Ras signaling and test the hypotheses that Raf is recruited to and activated in RANDs in a Ras-GTP dependent manner, that Raf is activated by H-Ras in dynamin-dependent RANDs and by K-Ras in actomyosin-driven RANDs, that Ras-PI3K signaling involves distinct RANDs from Ras-Raf signaling, and lastly, that the abundance of relevant RANDs determines the ability of Ras to activate Raf or PI3K or both. Together, these studies will yield detailed molecular insight into how Ras activities are regulated on the membrane to achieve functional specificity and diversity. Ras is often aberrantly activated in diseases such as cancer, and we anticipate the results to lend new strategies for manipulating Ras activity for therapeutic purposes.